[5]-Helistatins: Tubulin-Binding Helicenes with Antimitotic Activity

Helicenes are high interest synthetic targets with unique conjugated helical structures that have found important technological applications. Despite this interest, helicenes have had limited impact in chemical biology. Herein, we disclose a first-in-class antimitotic helicene, helistatin 1 (HA-1), where the helicene scaffold acts as a structural mimic of colchicine, a known antimitotic drug. The synthesis proceeds via sequential Pd-catalyzed coupling reactions and a π-Lewis acid cycloisomerization mediated by PtCl2. HA-1 was found to block microtubule polymerization in both cell-free and live cell assays. Not only does this demonstrate the feasibility of using helicenes as bioactive scaffolds against protein targets, but also suggests wider potential for the use of helicenes as isosteres of biaryls or cis-stilbenes—themselves common drug and natural product scaffolds. Overall, this study further supports future opportunities for helicenes for a range of chemical biological applications.


Molecular docking and predictive properties of [5]-helistatin, HA-1 Docking methodology
Docking studies were carried out with Flare, version 5, Cresset®, Litlington, Cambridgeshire, UK; http://www.cresset-group.com/flare/. [1][2][3] The crystal structure files (PDB: 1SAO and 4O2B) were downloaded directly from the protein data bank. Default protein preparation was carried out to set charge states and tautomers and to define the reference molecules (DAMAcolchicine and colchicine for 1SAO and 4O2B, respectively). The ligands CA4 and (P)-HA-1 were then imported and were subjected to energy minimisation to ascertain the most suitable conformers. These energy-minimised structures were then aligned to the reference molecules via molecular field and shape-guided substructure alignment. The pdb file for each ligand is provided as supplementary information (Files S7 to S10). The   Scheme S1 -Synthesis of phenol-functionalized helistatin was initially attempted via oxidative photocyclization methodology, based on conditions first reported by Liu and coworkers. 6 In the final photoisomerization step, MS and NMR analysis showed the formation of the furan, presumably via a 5-endo-trig cyclisation mediated by radical abstraction of the phenol. The bis-stilbene (i) was MOM-protected, however, only decomposition products were observed. The cause of this was believed to be a result of direct competition between the photoisomerization process and fluorescent relaxation of the molecule back to the ground state. Further attempts at forming this linkage in flow were also unsuccessful. This was likely due to the electronic mismatch between the aryl iodide and the electron neutral part of the phenanthrene. Several phenol protecting groups were examined (Bz, Bn, Tos), however, none of these afforded any cyclized product.  11 The coupled product (ii) was formed as a minor component in the reaction mixture, however, decomposition occurred during work-up and purification, suggesting that these aldehydes are particularly unstable. π Lewis-acid mediated cyclization of alkynes was tested in parallel and was found to be far more effective, therefore this route was not pursued any further.

General experimental
All reagents and solvents were purchased from commercial sources and used as supplied unless otherwise indicated. All reactions were carried out under an inert atmosphere, using anhydrous solvents. All reactions were monitored by thin-layer chromatography (TLC) using  All other experimental data was found to be in accordance with literature. 12 *In one instance a white precipitate was seen to form that soon dissolved upon warming the reaction flask to room temperature. **No evidence of the mono-silylated product was detected.
***On a smaller scale, during the course of reaction development, we noted a mixture of monoand bis-silylated product. Recrystallisation, as reported by Serwatowski and co-workers, 12 afforded pure bis-silylated product with significant loss of yield. However, the mono-silylated product can be efficiently distilled off (1 mbar vacuum pressure) leaving the bis-silylated product behind in the distillation flask. A Si plug (100% hexane) was then utilized to remove the majority of the coloured impurities. All other experimental data was found to be in accordance with literature. 13 Br Br I I 4 To a mixture of 3 (37.5 g, 77.2 mmol), CuI (1.46 g, 7.7 mmol) and PdCl2(PPh3)2 (2.70 g, 3.8 mmol) was added PhMe (100 mL) followed by (triisopropylsilyl)acetylene (36 mL, 160 mmol) and diisopropylamine (50 mL). The reaction mixture was heated to 70 ºC for 6 h.
Subsequently, the reaction was cooled to 0 ºC, diluted with Et2O and H2O before the phases were separated. The aqueous layer was extracted with Et2O and the combined organic layers washed with 1 M HCl, NaHCO3 (sat. aq.), brine and dried over anhydrous MgSO4 before being concentrated in vacuo. The crude product was purified using a Si plug (100% hexane) or, alternatively, through re-crystallisation from iPrOH. In both cases, the title compound was obtained as an off-white powder (71% yield, 32.6 g). The following procedure has been adapted from the literature. 15 To 8 (4.9 g, 16.0 mmol) was added MeOH (32 mL) and MeCN (32 mL) followed by a solution of KF (3.7 g, 63.8 mmol) in H2O (7 mL).  To a mixture of 5 (5.2 g, 7.8 mmol), 9 (2.9 g, 9.9 mmol), Cs2CO3 (7.

Purification of 3 with Cu(0) turnings
As previously noted, samples of 3 were found to be contaminated with elemental sulphur that impacted the subsequent Sonogashira coupling. Visual examination of contaminated batches of 3 revealed the presence of yellow crystals which were assumed to elemental sulphur. Upon treatment with Cu(0) turnings, as detailed above, the resultant material was found to be devoid of such yellow crystals and the Sonogashira coupling step proceeded smoothly. Furthermore, we extracted the yellow crystals from contaminated batches and submitted them for analysis by X-Ray crystallography (see Section 3.3; 3-contaminant), thus, confirming the presence of sulphur.

Chiral HPLC Purification of HA-1
A racemic sample of HA-1 was dissolved in neat iPrOH to yield a final sample concentration of ~2.5 mg/mL. Analytical HPLC runs were conducted using an Agilent 1260 LC equipped with a CHIRALPAK-IA column (

Figure S3
A chiral HPLC trace of racemic HA-1.

Figure S4
Extracted absorption spectra of the peaks at 8.0 min and 24.6 min, from the above HPLC trace, are shown on the left and right, respectively. The λmax is 240 nm.

X-Ray Crystallography
The X-ray crystal structure of 2 Crystal data for 2: C12H20Br2Si2, M = 380.28, monoclinic, P21/n (no. 14), a = 16.5998 (5) found to sit across a C2 axis that bisects the S1-S1A and S4-S4A bonds. were restrained to be similar, and only the non-hydrogen atoms of the major occupancy orientation were refined anisotropically (those of the minor occupancy orientation were refined isotropically).

2-A 2-B 8
We examined the enantiomerisation process of HA-1 at a biologically relevant temperature of 37.5 ºC. CD spectra of (P)-HA-1 and (M)-HA-1 are shown in Figure S6A. The enantiomerisation kinetics of (P)-HA-1 was also studied by CD spectroscopy (Figure S6B) to obtain a half-life of 1.6 h at 37.5 ºC. For comparison, pentahelicene ( Figure S6C) has an enantiomerisation half-life of 29 h at 25 ºC. 18 Utilising an Eyring plot (see Table S1 and Figure   S7), we extrapolated this literature data to obtain a half-life of 6 h at 37.5 ºC for the enantiomerisation of pentahelicene. The activation parameters for pentahelicene have been previously reported and are duplicated in Table S1. 19 Assuming that enantiomerisation is a first-order single-step process, the thermodynamic parameters can be determined using the Eyring Equation below:

HA-1
Where # ! is the rate constant of enantiomerisation, $ is the temperature, & is the transmission coefficient which is set to 0.5, 20 # " is the Boltzmann constant, ℎ is Planck's constant, ∆* ‡ is the activation entropy, + is the ideal gas constant and ∆- ‡ is the enthalpy of activation. Utilising the parameters in Table S1, the Eyring plot in Figure S7 is obtained. to measure metabolic activity (through reduction to fluorescent resorufin) as a readout for live cell count. 21 Fluorescence was measured at 590 nm (excitation 544 nm) using a FLUOstar Omega microplate reader (BMG Labtech). Absorbance data was normalized to viable cell count from the cosolvent control cells as 100% viability, where 0% viability was set to correspond to fluorescence signal from PBS with no cells, treated with resazurin (this underestimates true values corresponding to "no live cells" by ca. 5-15%, but does not affect assay outcomes and interpretation). Three independent experiments were performed. Viability data were plotted against the log of compound concentration (log10([drug]) (M)).
One representative HeLa experiment out of three is shown as Figure 3A (one datapoint per technical replicate). HA-1 shows antiproliferative activity while HA-2 does not.
Polymerisation was performed at 5 mg/mL tubulin, in polymerisation buffer BRB80 (80 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) pH = 6.9; 0.5 mM EGTA; 2 mM MgCl2), in a cuvette (120 μL final volume, 1 cm path length) in a Agilent CaryScan 60 with Peltier cell temperature control unit maintained at 37°C; with glycerol (10 µL). Tubulin was first incubated for 5 min at 37°C with the test compound in buffer with 3% DMSO, without GTP. Then GTP was added (1 µL spike, with mixing, final GTP concentration 1 mM) to initiate polymerisation, and the change in absorbance at 340 nm due to scattering from the turbid medium was monitored (greater turbidity = more polymerisation).

Cellular microtubule dynamics imaging
HeLa cells were transfected with EB3-GFP using FuGENE 6 (Promega) according to manufacturer's instructions. Experiments were imaged on a Nikon Eclipse Ti microscope